Summary: This research program is concerned with elucidation of the basic mechanisms of protective immunity to intracellular bacterial pathogens, including Mycobacterium tuberculosis, Francisella tularensis, and Listeria monocytogenes. A better understanding of the nature of protective immunity is essential to the rational design of new or improved vaccines, and prediction of useful correlates of protection. Thus we are characterizing the pathology of infection, the cell types involved, their cell-cell interactions and products, the specificity of the responses, the recognition receptors used, and the nature of the cellular responses provoked in the context of in vivo primary and secondary intracellular bacterial infection. Murine infection with Francisella tularensis Live Vaccine Strain (LVS), a gram negative facultative intracellular bacterium that replicates in macrophages, allows concurrent study of sublethal infection, lethal infection, and immune memory. Protective immune responses to F. tularensis also appear quite similar to those of M. tuberculosis (M. tb., TB) and L. monocytogenes. Thus each can be studied as representative of this class of pathogens, with a view toward identifying common or distinct patters of immune responses to intracellular bacteria. Studies this year have focused on several areas, including: the roles of T cells, B cells, IL12 and interferon gamma in resolution of primary and secondary LVS and M. tb. infection; early events, including the role of B cells, in establishment of lung pathology and dissemination of M. tuberculosis; and the role of recognition of bacterial DNA containing CpG motifs by the innate immune system in host response to LVS and M. tuberculosis. All these studies took advantage of a newly developed, novel in vitro culture system that replicates many of the features of in vivo infection. Bone marrow macrophages infected with LVS or M. tb. supported exponential growth of bacteria; addition of spleen or lung cells from LVS-immune or TB-immune mice specifically controlled LVS or M. tb. bacterial growth (respectively). This culture system therefore permits direct study of control of intracellular bacterial growth without any assumptions about the mechanisms of control. Using infected macrophages from interferon gamma receptor KO mice, we have demonstrated that there are significant IFN-indepedent mechanisms of immunity. These included contact-dependent mechanisms such as FasL, but not perforin, and contact-independent mechanisms such as elaboration of TNF alpha. In companion in vivo experiments, IL12 p40 knockout mice but not IL12 p35 knockout mice exhibited a chronic LVS infection in the absence of IL12 p40 and with minimal interferon gamma production. Bacteriostasis but not bacterial clearance is dependent not on interferon gamma; clearance is dependent on a previously unknown function of p40. In studies of early events in development of pathology and dissemination of M. tb., we are using transgenic mice that contain mature B cells but lack M cells as well as the ability to secrete antibodies to examine the relative contributions of each to M. tb. lung pathology. In studies on protection provided by DNA containing CpG motifs, we demonstrated that control of intracellular bacterial growth by DNA-primed lymphocytes is dependent entirely on soluble mediators, including interferon gamma, IL12, and TNF alpha. While natural killer cells are activated by CpG DNA and produce interferon gamma, NK cells are not absolutely required for in vivo protection. Further understanding of such immunobiological properties of bacterial DNA will be important in evaluation of DNA vaccines, the use of bacterial DNA as an adjuvant, and therapeutic applications of bacterial DNA.